Project Summary/Abstract: During gametogenesis, an exogenous signal initiates cells to enter meiosis and differentiate into gametes. A failure to maintain the meiotic process can result in inappropriate cell proliferation, genome damage, and developmental defects. Despite the importance of maintaining meiosis, there is a lack of a molecular understanding of how external signals influence the cell-cycle regulatory network to prevent cells from inappropriately exiting meiosis. The objective of this proposal is to determine how cell-cycle regulatory networks, influenced by external signals, maintain the meiotic or mitotic cell identity. Due to the difficult in manipulating the external environment of meiotic cells in multi-cellular eukaryotic organisms, these studies employ S. cerevisiae as the model organism. Microfluidic tools have been developed to temporally and precisely manipulate the cells' external signals, monitor the output of the intracellular signaling pathways, and identify cell cycle outcomes in individual cells. Thes innovations allow the investigation of how cells integrate signals to maintain meiosis, how proteins function within meiotic networks, and how checkpoint mechanisms affect cell-cycle outcomes. The rationale for the proposed research is that the questions chosen are focused on processes that are likely to be highly conserved, allowing the findings in budding yeast to uncover general mechanisms of cell-cycle control. Strong preliminary data has guided the following three specific aims: 1) Determine the regulatory mechanisms that govern meiotic commitment; 2) Determine the role of meiotic checkpoint pathways in coordinating meiotic commitment and regulating the return to mitosis; and, 3) Identify and analyze cell-cycle regulators that control the crucial G2/M checkpoint during the return to mitosis. In the first aim, the microfluidic tools will be implemented to test the hypothesis that a feedback pathway establishes the commitment to maintain meiosis. Moreover, cell-cycle regulators will be tested for their role in meiotic maintenance. The second aim tests the hypothesis that meiotic checkpoint mechanisms coordinate the temporal regulation of the commitment to maintain meiosis. The third aim will employ biochemical techniques and a genetic screen to determine how the highly conserved G2/M checkpoint coordinates cell cycle events in cells that exit meiosis and return to mitosis. The innovative approach of using microfluidics to manipulate the application and withdrawal of an external signal while monitoring single cells allows the testing of novel hypotheses about cell-cycle regulation in response to external signals. The proposed research is significant because the results are expected to uncover general principles of cell-cycle regulation that protect genome integrity by maintaining the meiotic or mitotic cell identity. Ultimately, the results will further our understanding of how errors in this process facilitate neoplasia and developmental abnormalities.